RESUMEN
X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened â¼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.
Asunto(s)
Aurora Quinasas/antagonistas & inhibidores , Metilación de ADN/efectos de los fármacos , Inactivación del Cromosoma X/efectos de los fármacos , Animales , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/genética , Aurora Quinasa B/antagonistas & inhibidores , Aurora Quinasa B/genética , Aurora Quinasas/genética , Azacitidina/administración & dosificación , Azacitidina/análogos & derivados , Azepinas/administración & dosificación , Línea Celular , Decitabina , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Femenino , Técnicas de Silenciamiento del Gen , Genes Ligados a X , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ensayos Analíticos de Alto Rendimiento , Ratones , Ratones Transgénicos , Piperazinas/administración & dosificación , Pirimidinas/administración & dosificación , Cromosoma X/efectos de los fármacos , Cromosoma X/genéticaRESUMEN
A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D(1), known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds.